PROJECT SUMMARY Formation of a functional musculoskeletal system is dependent on the integration of contractile muscle to load-bearing tendon to bone. These tissues are initially specified independently of each other, but then seamlessly integrate as development progresses. Researchers investigating the mechanisms behind musculoskeletal assembly have primarily focused on the signaling pathways that regulate cellular behavior. The current paradigm is that the interfaces between muscle and tendon and bone are established through cell ? cell interactions and reciprocal signaling between the separate tissues via secreted signals. However, preliminary studies by my lab indicate that an extracellular matrix (ECM)-based template, deposited before the formation of these interfaces, may also direct the proper combinations between muscle and tendon and bone. The role of the ECM during early musculoskeletal development has been largely overlooked due to a lack of tools. To address these challenges, my laboratory has been developing novel methods to map the composition and organization of the ECM over the course of musculoskeletal development. Recently, we demonstrated that the murine embryo could be labeled with non-canonical amino acids (ncAAs), which are powerful biochemical tools that enable the visualization, enrichment and identification of newly synthesized proteins within complex mixtures. In addition, my lab has pioneered optical clearing methods to image the 3D ECM architecture of biomarkers deep within developing and adult musculoskeletal tissues. The goal of this project is to identify the composition of the ECM template and the mechanisms by which the template becomes aligned and adapts to growth. Established knockout models in which musculoskeletal patterning is disrupted will be used to test the hypothesis that an ECM-based template, specified before tendon and muscle differentiation, is necessary for the alignment and correct integration of load-bearing muscles and tendons during vertebrate forelimb development. Downstream target(s) that regulate template formation and remodeling will be identified via mass spectrometry combined with ncAA labeling. The spatio-temporal expression of candidate molecules will be visualized with respect to the template and connective tissue cells using clearing techniques developed within my laboratory. The strongest candidates will be knocked down in a tissue specific manner and severity of phenotype will be quantified using 3D morphological measurements. Template extensibility will be measured by time-lapse imaging of embryos cultured ex vivo and quantifying network deformation. Successful completion of the proposed studies will identify the composition and spatial distribution of the ECM that provide the foundation for musculoskeletal architecture, reveal how muscles and tendons are aligned and provide insight into ECM mechanics in vivo. Together, these results will provide design parameters for regenerative scaffolds that can successfully restore functionality to damaged musculoskeletal tissues.